μXRF and LA-ICP-TQMS for quantitative bioimaging of iron in organ samples of a hemochromatosis model.

Hereditary hemochromatosis is the most common autosomal recessive genetic disorder of the iron metabolism. Iron accumulation in various organs, especially in liver and pancreas leads to diseases and may cause organ failure. In this study, methods for elemental bioimaging by means of quantitative micro X-ray fluorescence analysis (μXRF) and laser ablation-inductively coupled plasma-triple quadrupole mass spectrometry (LA-ICP-TQMS) were developed and applied to investigate the pathophysiological development of iron accumulation in murine tissue based on animals with an iron-overload phenotype caused by a hepatocyte-specific genetic mutation. The use of an external calibration with matrix-matched gelatin standards enables the quantification of iron by means of μXRF without the typically used fundamental parameters method or Monte Carlo simulation, which becomes more imprecise when analyzing thin tissue sections. A fast, non-destructive screening of the iron concentration and distribution with a spatial resolution of 25 μm in liver samples of iron-overload mice was developed. For improved limits of detection and higher spatial resolution down to 4 μm, LA-ICP-TQMS was used with oxygen as reaction gas. By monitoring the mass shift of 56 Fe to 56 Fe16 O, a limit of detection of 0.5 μg/g was obtained. With this method, liver and pancreas samples of iron-overload mice as well as control mice were successfully analyzed. The high spatial resolution enabled the analysis of the iron distribution in different liver lobules. Compared to the established Prussian blue staining, both developed methods proved to be superior due to the possibility of direct iron quantification in the tissues.